| Objective:To study the role of IL-10 in GVHD by upregulation IL-10 of Naive CD4+T cells treated with Galectin-1 on bone marrow derived dendritic cells(BM-DCgal).Methods:(1) Bone marrow progenitor cells were prepared from BALB/c mice and stimulated by GM-CSF and IL-4 to generate DC, Different doses of Galectin-1 Were added.(2) Galectin-1 treatmented cells and dendritic cells were sorted by flow cytometry.(3) Spleen lymphocytes were prepared from C57BL/6 mice.(4) Naive CD4+T cells were sorted by MACS.(5) Naive CD4+T cells were co-cultured with BM-DC or BM-DCgal sorted by flow cytometry,and treated with IL-10R(IL-10 receptor) antibody.(6) ELISA method was used to analyze the secretion of IL-10 in BM-DC and BM-DCgal maturation.(7) The expression of IL-10 in the BM-DC and BM-DCgal co-cultural cells was detected by Real time-PCR.(8) The cytokines secretion of IL-17A, IL-4, IL-10, IFN-y by ELISA and the cell surface marker of IL-17A, IL-10, IFN-y by flow cytometry were compared.Results:(1) The dendritic cells were prepared and cultured,as well as were sorted successfully.(2) Naive CD4+T cells were successfully sorted by MACS.(3) The expression of IL-10 in the BM-DCgal co-cultural cells is higher than BM-DC co-cultural cells by quantitative real-time PCR.(4) BM-DCgal co-cultural cells have higher IL-10 expression and less IL-17A, IFN-γ expression than BM-DC co-cultural cells.(5) BM-DCgal co-cultural cells treated by IL-10R antibody have less IL-10 expression and higher IL-17A, IFN-y expression cells..(6) BM-DCgal co-cultural system has higher IL-10 secretion than BM-DC co-cultural system by ELISA.(7) BM-DCgal co-cultural system with IL-10R antibody has less IL-10 secretion than BM-DC co-cultural system wtih IL-10R antibody by ELISA.Conclusions:This study showed that the expression of IL-10 on CD4+T cells which co-cultured with BM-DCgal increased. IL-10 may play an important role in GVHD by promoting naive CD4+T cells treated by BM-DCgal differention into Trl cells. |
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